1. Field of the Invention
This invention relates generally to antimicrobial peptides and specifically to antimicrobial cationic peptides useful for overcoming antibiotic resistance and effective as therapeutics for pathologies resulting from microbial infections.
2. Description of Related Art
In 1981, the self-promoted uptake hypothesis was first proposed to explain the mechanism of action of polycationic antibiotics in Pseudomonas aeruginosa. According to this hypothesis, polycations interact with sites on the outer membranes of Gram-negative bacteria at which divalent cations cross-bridge adjacent lipopolysaccharide molecules. Due to their higher affinity for these sites, polycations displace the divalent cations and, since the polycations are bulkier than the divalent cations, cause structural perturbations in the outer membrane. These perturbations result in increased outer membrane permeability to compounds such as the xcex2-lactam antibiotic nitrocefin, the eukaryotic non-specific defense protein lysozyme and to hydrophobic substances. By analogy, molecules accessing this pathway are proposed to promote their own uptake.
It has been clearly demonstrated that the outer membranes of Gram-negative bacteria are semipermeable molecular Asieves≅ which restrict access of antibiotics and host defense molecules to their targets within the bacterial cell. Thus, cations and polycations which access the self-promoted uptake system are, by virtue of their ability to interact with and break down the outer membrane permeability barrier, capable of increasing the susceptibility of Gram-negative pathogenic bacteria to antibiotics and host defense molecules. Hancock and Wong demonstrated that a broad range of such compounds could overcome the permeability barrier and coined the name Apermeabilizers≅ to describe them (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984). While self-promoted uptake and permeabilizers were first described for P. aeruginosa, they have now been described for a variety of Gram-negative bacteria.
Over the past decade, non-specific defense molecules have been described in many animals, including insects and humans. One subset of these molecules have in common the following features: (a) they are small peptides, usually 15-35 amino acids in length, (b) they contain 4 or more positively charged amino acid residues, either lysines or arginines, and (c) they are found in high abundance in the organisms from which they derive. Several of these molecules have been isolated, amino acid sequenced and described in the patent literature (e.g., cecropins: WO8900199, WO 8805826, WO8604356, WO 8805826; defensins: EP 193351, EP 85250, EP 162161, U.S. Pat. No. 4,659,692, WO 8911291). However, only limited amounts of these peptides can be isolated from the host species. For example, Sawyer, et al., (Infect. Immun. 56:693, 1988) isolated 100-200 mg of rabbit neutrophil defensins 1 and 2 from 109 primed peritoneal neutrophils or lipopolysaccharide-elicited alveolar macrophages (i.e., the numbers present in a whole animal).
Some cationic antibacterial peptides are of relatively high molecular weight (greater than about 25 kDa) and are effective against certain Gram negative bacteria such as Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa by damaging the cytoplasmic membrane leading to increased membrane permeability. Human bactericidal/permeability increasing protein (BPI) is a strongly basic protein with a molecular weight of about 59 kDa. It is believed that, when bound to outer membrane of the susceptible bacterial cells, hydrophobic channels through the outer envelope are exposed, and as a secondary effect, there is a selective activation of autolytic enzymes including phospholipase and peptidoglycan hydrolases. Gram positive bacteria, certain Gram negative bacteria and fungi are not affected by BPI in vitro.
Low molecular weight cationic peptides (10 kDa to 25 kDa) have been reported which inhibit the growth of such Gram positive bacteria as Staphylococcus aureus (Root and Cohen, Rev. Infect. Dis., 3:565-598, 1981). In addition cationic peptides with fungicidal activity have been identified in alveolar macrophages. It is believed that cationic peptides are most efficient in killing phagocytized microorganisms in combination with other microbicidal defense mechanisms.
Generally defensins are relatively small polypeptides of about 3-4 kDa, rich in cysteine and arginine. Gabay et al. (Proc. Natl. Acad. Sci. USA, 86:5610-5614, 1989) used reversed phase HPLC to purify 12 major polypeptides from the azurophil granules of human polymorphonuclear leukocytes (PMNs). Defensins as a class have activity against some bacteria, fungi and viruses. The defensins are believed to have a molecular structure stabilized by cysteine infrastructure, which are essential for biological activity. The gene for human defensin has been cloned and sequenced, but no successful expression has been demonstrated, as yet. Furthermore, production of these peptides using peptide synthesis technology produces peptides in limited amounts and is expensive when scaled up or when many variant peptides must be produced. Also, structural analysis is difficult without specific incorporation of 15N and 13C tagged amino acids which is prohibitively expensive using amino acid synthesis technology.
Another class of antimicrobial peptides are those known as magainins and at least five of which can be isolated from the African clawed frog (Xenopus laevis). The natural proteins are active against a broad range of microorganisms including bacteria, fungi and protozoans (Zasloff, Proc. Natl. Acad. Sci., USA, 84:5499, 1987). The broad spectrum antimicrobial activity is present in synthetic peptides and in certain truncated analogs of the natural proteins. Derivatives of about 19 to about 23 amino acids have antibacterial activity as measured using Escherichia coli. In addition, the antimicrobial activity of magainin appears to result in the disruption of the membrane functions of Paramecium caudatum. The configurations of the bioactive peptides can be modeled as amphophilic alpha-helices and are sufficiently long to span a lipid bilayer (Zasloff et al., Proc. Natl. Acad. Sci., USA, 85:910, 1988). Spanning a lipid bilayer is believed to require at least twenty amino acid residues in an alpha-helical configuration (Kaiser, Ann. Rev. Biophys. Chem., 16:562, 1987)
Cationic peptides containing a disulphide bond forming a looped structure were recently identified (Morikawa et al., Biochim. Biophys. Res. Commun. 189:184, 1992; Simmaco et al., FEBS 324:159, 1993; Clark et al., J. Biol. Chem. 269:10849, 1994). One member of this group, bactenecin (i.e., dodecapeptide), is a twelve amino acid peptide isolated from bovine neutrophils (Romeo et al., J. Biol. Chem. 263:9573, 1988). Bactenecin is the smallest known cationic antimicrobial peptide. Two cysteine residues form a disulphide bond to make bactenecin a loop molecule. Bactenecin was previously found to be active against Escherischia coli and Staphylococcus aureus, and strongly cytotoxic for rat embryonic neurons, fetal rat astrocytes and human glioblastoma cells (Radermacher et al., J. Neuroscience Res. 36:657, 1993).
Synthetic peptide chemistry has determined that xcex1-helices are a common structural motif found in both antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Cecropins were initially discovered in insects but later found in other animals including mammals. Electron microscopy has revealed that cecropin-induced inhibition of bacterial growth is due, in part, to bacterial wall lysis. Resistance to such a generally destructive mechanism may prove difficult for some microbial pathogens, as compared with the more specific mechanisms of the currently used antibiotics. Further, the bee venom peptide melittin is known to form channel-like structures in biological membranes and retrains pharmacological properties in intact tissues including hemolysis, cytolysis, contractures of muscle, membrane depolarization and activation of tissue phospholipase C.
There is a need to develop peptides having a broad range of potent antimicrobial activity against a plurality of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, parasites, viruses and the like. The identification of novel antimicrobial cationic peptides which overcome antibiotic resistance and are effective as therapeutics for microbial pathogens would aid in combating such organisms.
The present invention provides antimicrobial cationic peptides effective for inhibiting the growth of a plurality of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, parasites and viruses. Exemplary peptides include:
and analogs, derivatives, amidated variations and conservative variations thereof.
The invention also provides a method of inhibiting the growth of bacteria including contacting the bacteria with an inhibiting effective amount of at least one peptide of the invention alone, or in combination with at least antibiotic. Classes of antibiotics that can be used in synergistic therapy with the peptides of the invention include aminoglycoside, penicillin, cephalosporine, fluoroquinolone, carbepenem, tetracycline and macrolides.
The invention further provides polynucleotides that encode the peptides of SEQ ID NOs:3-12 and 14-21 of the invention. In one aspect, amidated derivatives of antimicrobial peptides encoded by such polynucleotides are included in the invention.
In another embodiment, the invention provides a method of inhibiting an endotoxemia- or sepsis-associated disorder in a subject having or at risk of having such a disorder, by administering to the subject a therapeutically effective amount of at least one peptide of the invention.
The invention also provides a method of inhibiting the growth of a eukaryotic cell. The method includes contacting the cell with an inhibiting effective amount of a peptide or combination thereof of the invention, alone, or in combination with an agent effective for inhibiting eukaryotic cell growth. Such agents which can be used for synergistic therapy with the peptides of the invention include bleomycin, neocarcinostatin, suramin, doxorubicin, taxol, mitomycin C and cisplatin.
The invention further provides a method of inhibiting a cell proliferation-associated disorder in a subject having or at risk of having such a disorder. The method includes administering to the subject a therapeutically effective amount of at least one peptide of the invention, alone, or in combination with an agent effective for inhibiting eukaryotic cell growth. Such agents which can be used for synergistic therapy with the peptides of the invention include bleomycin, neocarcinostatin, suramin, doxorubicin, taxol, mitomycin C and cisplatin.
The invention further provides a method for accelerating wound healing in a subject in need of such treatment including contacting the site of the wound with a therapeutically effective amount of a composition containing an antimicrobial cationic peptide of the invention.
In another embodiment, the invention provides a method of treating a respiratory or pulmonary associated disorder in a subject having or at risk of having such a disorder, comprising administering to the subject a therapeutically effective amount of peptide.
In another embodiment, the invention provides a transgenic non-human animal expressing an antimicrobial cationic peptide of the invention.
In yet another embodiment the invention provides transgenic fish expressing an antimicrobial cationic peptide of the invention, a method for producing such a transgenic fish and a method for detecting expression of a transgene in a transgenic fish of the invention.